Evaluation of cell viability is important for assessing the effect of drugs, environmental pollutants, irradiation, temperature, ionic extremes, and other potential biological modifiers. Traditionally, cell membrane integrity is used as an indicator of cell viability, as damage to the protective cell membrane often results in loss of cell structure, leakage of critical intracellular contents, breakdown of essential ionic gradients and ultimately cell death. Another indicator of cell viability is intracellular activity, the presence of which activity indicates that the cell is able to metabolize, grow, reproduce, maintain electrical membrane potential, or perform some other cell function critical for viability. Conversely, the lack of such activity is often used as an indicator of cell death.
Although a single dye can be used to assess viability, the use of a combination of dyes has advantages. First, the use of a dye combination allows the investigator to determine the ratio of the number of cells that show a response to the one dye versus the total number of cells or versus those cells that do not respond. In addition, the second dye can be used as a positive control to indicate that other cells are present that did not stain with the first dye. For this reason, methods of determining viability that use a combination of dyes are generally preferred.
Several methods using a combination of fluorescent dyes for the analysis of cell viability have been developed, including methods that use differential fluorescent staining of live and dead cells. Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS Sets 25 & 31 (1992) (incorporated by reference). Live, intact cells can generally be distinguished from dead cells with compromised membranes by differential staining using a cell-impermeant fluorescent dye that only enters dead cells, and a cell-permeant dye that enters both live and dead cells but requires intracellular activity indicative of viability for the production of fluorescence. Alternatively, differential fluorescent staining can involve the use of two cell-permeant dyes where one stains both live and dead cells and the other stains cells only when an intracellular reaction produces fluorescence.
Not all fluorescent compounds that have these general characteristics are equally useful, however. A number of fluorescent viability stains, for example, have spectral or physical properties that are incompatible with available instrumentation or limit their use in conjunction with other materials. For instance, the use of acridine orange (U.S. Pat. No. 4,190,328) is severely limited because of high background signal and low fluorescence enhancement upon binding to nucleic acids (about two-fold). Nucleic acids complexed with DAPI or Hoechst 3342 are only excitable with UV light, which is incompatible with some instrumentation. More importantly, the spectral properties of DAPI- or Hoechst 33342-bound DNA overlap significantly with cellular autofluorescence.
Moreover, it is not uncommon that fluorescent materials that are useful with one type of cell, or will not produce the desired reaction in different types of cells. For example, calcein-AM and ethidium homodimer (Ser. No. 07/783,182 (filed Oct. 26, 1991) to Haugland et al.) now U.S. Pat. No. 5,314,805, have been found to be less useful with cells from prokaryotic microbes than with those of eukaryotic microbes (Kaneshiro, et al., J. MICROBIOLOG. METHODS 17, 1 (1993)).
The method of the present invention provides significant advantages over conventional methods. This method, which allows the determination of cell viability either simultaneously or sequentially, is applicable to a wide range of cells, regardless of the source. In addition, the availability of a family of dyes with similar permeability characteristics but different spectral characteristics allows the selection of complementary pairs impermeant and permeant stains. Furthermore, the method is extremely sensitive, reliable and fast, requiring no harsh reagents or special culturing conditions. It is useful for laboratory analysis, industrial process monitoring and environmental sampling.
The method of the invention utilizes a fluorogenic dye from a new family of unsymmetrical cyanine dyes that was unexpectedly found to label all cells tested, whether living or dead, described in copending applications Ser. No. 08/090,890, filed Jul. 12, 1993, now U.S. Pat. No. 5,436,134, by Haugland, et al. and FLUORESCENT ASSAY FOR BACTERIAL GRAM REACTION, filed Nov. 1, 1993 by Roth, et al.; both of which are continuations-in-part of patent application Ser. No. 08/047,683, filed Apr. 13, 1993, now abandoned, by Roth et al. (all three of which are incorporated by reference). Although certain unsymmetrical cyanine dyes were first described before the genetic role of nucleic acids was established (Brooker, et al., J. Am. Chem. Soc. 64, 199 (1942)), a variety of unsymmetrical cyanine dyes have now been found to be very effective in the fluorescent staining of DNA and RNA. U.S. Pat. Nos. 4,554,546 (to Wang, et al. 1985) and 5,057,413 (to Terstappen et al. 1991) disclose use of similar derivatives of thioflavins as nucleic acid stains. U.S. Pat. No. 4,937,198 (to Lee et al. 1990) discloses a fluorescent nucleic acid stain that preferentially stains the nucleic acids of bloodborne parasites with little staining of nucleated blood cells.
Closely related lower alkyl (1-6 carbons) substituted unsymmetrical cyanine dyes, exemplified by thiazole orange, are disclosed in U.S. Pat. No. 4,883,867, as having particular advantages in reticulocyte analysis. The attachment of bulkier (e.g. cyclic) structures to the parent unsymmetrical cyanine dye results in a number of unexpected advantages for this family of dyes. For example, although bulkier, the new dyes more quickly penetrate the cell membranes of a wider variety of cell types, including both Gram-positive and Gram-negative bacteria, as well as a variety of eukaryotic cells. Direct comparison of the rate of uptake with known dyes such as thiazole orange and its alkylated derivatives, shows enhanced uptake of the new compounds (FIG. 1). Moreover, a wider range of cells stained with the novel dyes generally exhibit much more fluorescence than cells stained with thiazole orange (Table 1), and the quantum yield of these new dyes is unexpectedly better than that of thiazole orange (Table 2). Furthermore, by simple synthetic modification, a family of dyes having absorption and emission spectral properties that cover most of the visible and near-infrared spectrum can be prepared. These features overcome the limitations imposed by thiazole orange and other unsymmetrical cyanine dyes for staining the nucleic acids of a wide variety of cells. The superior properties exhibited by these dyes were neither anticipated nor obvious in view of the known unsymmetrical cyanine dyes.
TABLE 1 __________________________________________________________________________ Fluorescence/Cell (ex 485/em 530).sup.1 Dye.sup.2 Thiazole Sample.sup.3 61 63 613 619 624 628 591 634 Orange __________________________________________________________________________ B. cereus 196 93 242 827 709 613 58 154 51 M. luteus 49 33 75 149 162 149 375 58 21 S. pyogenes 0.01 0.01 0.02 0.06 0.05 0.04 &lt;0.01 0.03 &lt;0.01 S. aureus 15 5 10 47 44 34 3 15 3.2 E. coli 10 6 12 26 24 28 2 4 2 S. oranienburg 10 4 10 18 15 19 2 5 1 K. pneumonia 10 5 10 12 17 20 2 4 3 S. sonnei 6 3 6 13 11 16 1 4 1 P. aeruginosa 5 3 6 16 14 14 1 3 2 __________________________________________________________________________ 1. Measured in a fluorescence microtiter plate reader with extraction and emission filters at 485+/-10 and 530+/-12, respectively. Fluorescence dat are corrected for cell number; but are not corrected for cell volume or nucleic acid content. 2. Optimal dye concentrations determined as in Example 3. 3. Suspension concentrations as used for Example 3.